Chaperoning heat shock proteins: Proteomic analysis and relevance for normal and dystrophin-deficient muscle

Molecular chaperones play a key role in normal muscle function and during physiological adaptations to extensive exercise and numerous forms of cellular stress. The various classes of HSPs and related chaperones are also involved in the molecular pathogenesis of a large number of neuromuscular diseases. Several MS-based proteomic studies have recently shown that the expression levels of molecular chaperones are severely altered in dystrophin-deficient muscles. Dystrophin isoform Dp427 (where Dp427 is dystrophin protein of 427 kDa) is a large membrane cytoskeletal protein and its deficiency is the primary underlying cause of Duchenne muscular dystrophy. Current efforts have focused on the establishment of a comprehensive biomarker signature of dystrophinopathy in order to improve diagnostic methods, establish reliable prognostic factors and identify novel therapeutic targets. Following an introduction into the biology of HSPs and their general role in skeletal muscle, this review outlines the proteomic profiling of molecular chaperones in dystrophinopathy. The focus is especially on the molecular fate of HSPs cardiovascular HSP (HSPB7), αBC (HSPB5), HSP70 (HSPA) and HSP90 (HSPC) in dystrophin-deficient muscles and their involvement in progressive muscular dystrophy. Furthermore, the potential usage of distinct chaperones as disease markers of secondary pathobiochemical changes for the evaluation of novel treatment options is discussed.     

Proteomic profile of differentially expressed plasma proteins from dystrophic mice and following suberoylanilide hydroxamic acid treatment

Purpose: Histone Deacetylase Inhibitors (DI) ameliorates dystrophic muscle regeneration restoring muscular strength in the mdx mouse model of Duchenne muscular dystrophy (DMD). The further development of these compounds as drugs for DMD treatment is currently hampered by the lack of knowledge about DIs effect in large dystrophic animal models and that of suitable biomarkers to monitor their efficacy. Experimental design: In this study we applied proteomic analysis to identify differentially expressed proteins present in plasma samples from mdx mice treated with the Suberoylanilide hydroxamic acid (SAHA) and relative normal controls (WT). Results: Several differentially expressed proteins were identified between untreated wild type and mdx mice. Among these, fibrinogen, epidermal growth factor 2 receptor, major urinary protein and glutathione peroxidase 3 (GPX3) were constitutively up‐regulated in mdx, while complement C3, complement C6, gelsolin, leukaemia inhibitory factor receptor (LIFr), and alpha 2 macroglobulin were down‐regulated compared to WT mice. SAHA determined the normalization of LIFr and GPX3 protein level while apoliprotein E was de novo up‐regulated in comparison to vehicle‐treated mdx mice. Conclusions and clinical relevance: Collectively, these data unravel potential serological disease biomarkers of mdx that could be useful to monitor muscular dystrophy response to DI treatment.     

The secretome of the working human skeletal muscle—A promising opportunity to combat the metabolic disaster?

Recent years have provided clear evidence for the skeletal muscle as an endocrine organ. Muscle contraction during physical activity has emerged as an important activator of the release of the proteins and peptides called “myokines." Diverse proteomic profiling approaches were applied to rodent and human skeletal muscle cells to characterize the complete secretome, to study the regulation of the secretome during cell differentiation or the release of myokines upon contractile activity of myotubes. Several of the exercise-regulated factors have the potency to mediate an interorgan crosstalk. The paracrine function of the secreted peptides and proteins to regulate muscle regeneration, tissue remodeling, and trainability can have direct effects on whole-body glucose disposal and oxygen consumption. The overall composition and dynamic of the myokinome are still incompletely characterized. Recent advantages in metabolomics and lipidomics will add metabolites and lipids with autocrine, paracrine, or endocrine function to the contraction-induced secretome of the skeletal muscle. The identification of these metabolites will lead to a more comprehensive view described by a new myo(metabo)kinome consisting of peptides, proteins, and metabolites.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

Profiling the potential biomarkers for cell differentiation of pancreatic cancer using iTRAQ and 2-D LC-MS/MS

Pancreatic cancer is a highly lethal disease that is difficult to diagnose at early stage and even more difficult to cure. SW1990 and PANC-1 represent the two cancer cell lines, which are both derived from pancreatic duct, but at different cell differentiation stages. In this study, we applied the iTRAQ-labeling technology and 2-D strong cation exchange/reversed phase liquid chromatography – LC-MS/MS) to profile the secreted proteins of SW1990 and PANC-1 cells in a conditioned cell culture medium. A total of 401 proteins were identified by MS/MS and protein database searching, the percentages of these proteins predicted in the categories of plasma membrane, intracellular and secreted proteins were 29.2, 32.7 and 38.2%, respectively. Fifty six proteins were identified with unknown functions and 19 proteins were quantified with significant level changes between the two cancer cell lines under the specific cell condition with 12 proteins being up-regulated (>1.3-fold change) in PANC-1 (e.g. FLJ31222 protein, 97 kDa protein, type IV collagenase precursor, 38 kDa protein and centaurin) and seven proteins being up-regulated in SW1990 (e.g. fibroblast growth factor receptor substrate 2, putative p150, hypothetical protein LOC 654463 and LOC 55701). The proteins with significant level changes may provide a baseline to investigate mechanisms underlying the differentiation of two cell lines and can be further screened for better protein biomarkers in pancreatic cancer.     

A windows software for simulation of the membrane excitation in nerve and skeletal muscle fiber.

A windows 95/98 software program for simulating membrane excitation in nerve and skeletal muscle fiber has been developed. This program simulates (1) the action potentials of the nerve under two conditions (space clamped conditions and conducting conditions), (2) the membrane currents of the nerve under voltage clamped conditions, and (3) the propagated action potentials of skeletal muscle fiber. Since users can utilize quadruple precision in the simulation of propagated action potentials, such simulation can be done for a long period (60 ms in skeletal muscle at 2 degrees C). In addition, users can change the conditions such as the capacitance of the membrane, the maximum conductance of the channel, etc., arbitrarily without quitting the program. It is also possible to easily modify the time and the voltage dependence of the gate particles in order to confirm the effects of various changes on the action potential or the membrane current. This program also has an analysis mode in which the current-voltage relationship of a channel can be measured automatically.     

An integrated intelligent computing model for the interpretation of EMG based neuromuscular diseases

Intelligent computing system (ICS) and knowledge-based system (KBS) have been widely used in the detection and interpretation of EMG (electromyography) based diseases. Heuristic-based detection methods of EMG parameters for a particular disease have also been reported in the literature but little effort has been made by researchers to combine rule-based reasoning (RBR) and case-based reasoning of KBS, and ANN (artificial neural nets) of ICS. Integrating the methods in KBS and ICS improves the computational and reasoning efficiency of the problem-solving strategy. We have developed an integrated model of CBR and RBR for generating cases, and ANN for matching cases for the interpretation and diagnosis of neuromuscular diseases. We have hierarchically structured the neuromuscular diseases in terms of their physio-pyscho (muscular, cognitive and psychological) parameters and EMG based parameters (amplitude, duration, phase etc.). Cumulative confidence factor is computed at different node from lowest to highest level of hierarchal structure in the process of diagnosis of the neuromuscular diseases. The diseases considered are Duchenne muscular dystrophy, Polymyostits, Endocrine myopathy, Metabolic myopathy, Neuropathy, Poliomyletis and Myasthenia gravis. The basic objective of this work is to develop an integrated model of RBR, CBR and ANN in which RBR is used to hierarchically correlate the sign and symptom of the disease and also to compute cumulative confidence factor (CCF) of the diseases. CBR is used for diagnosing the neuromuscular diseases and to find the relative importance of sign and symptoms of a diseases to other diseases and ANN is used for matching process in CBR.     

生命-机电系统融合的类生命机器人

类生命机器人是一种由生命系统与机电系统在分子、细胞、组织尺度有机融合而成的新型机器人,具有能量转换效率高、续航时间长、小型化等潜在优点,可为突破传统机器人在驱动、感知、智能等方面所面临的瓶颈提供新的思路。本文围绕类生命机器人的研究现状,首先根据不同的生命材料（如心肌细胞、骨骼肌细胞、神经-肌肉、昆虫肌肉组织、微生物）对现有类生命机器人进行了系统性的归类与介绍。然后,对类生命机器人常用的非生命材料（如聚二甲基硅氧烷（PDMS）、水凝胶）、控制方法（如电控制、光控制、化学控制、磁控制）及其应用场景（如微型机器人、芯片实验室、生物医学）进行了详细的讨论。最后,对类生命机器人研究所面临的潜在挑战与可能的发展方向进行了分析与展望。     

Exploration of the normal human bronchoalveolar lavage fluid proteome

We obtained insight into normal lung function by proteome analysis of bronchoalveolar lavage fluid (BALF) from six normal human subjects using a "Lyse-N-Go' shotgun proteomic protocol. Intra-sample variation was calculated using three different label-free methods, (i) protein sequence coverage; (ii) peptide spectral counts and (iii) peptide single-ion current areas (PICA), which generates protein expression data by summation of the area under the curve for a given peptide single-ion current trace and then adding values for all peptides from that same parent protein. PICA gave the least intra-subject variability and was used to calculate differences in protein expression between the six subjects. We observed an average threefold inter-sample variability, which affects analysis of changes in protein expression that occur in different diseases. We detected 167 unique proteins with >100 proteins detected in each of the six individual BAL samples, 42 of which were common to all six subjects. Gene ontology analysis demonstrated enrichment of several biological processes in the lung, reflecting its expected role in gas exchange and host defense as an immune organ. The same biological processes were enriched compared to either plasma or total genome proteome, suggesting an active enrichment of plasma proteins in the lung rather than passive capillary leak.     

Proteomics cataloging analysis of human expressed prostatic secretions reveals rich source of biomarker candidates

Expressed prostatic secretions (EPS) contain proteins of prostate origin that may reflect the health status of the prostate and be used as diagnostic markers for prostate diseases including prostatitis, benign prostatic hyperplasia, and prostate cancer. Despite their importance and potential applications, a complete catalog of EPS proteins is not yet available. We, therefore, undertook a comprehensive analysis of the EPS proteome using 2‐D micro‐LC combined with MS/MS. Using stringent filtering criteria, we identified a list of 114 proteins with at least two unique‐peptide hits and an additional 75 proteins with only a single unique‐peptide hit. The proteins identified include kallikrein 2 (KLK2), KLK3 (prostate‐specific antigen), KLK11, and nine cluster of differentiation (CD) molecules including CD10, CD13, CD14, CD26, CD66a, CD66c, CD 143, CD177, and CD224. To our knowledge, this list represents the first comprehensive characterization of the EPS proteome, and it provides a candidate biomarker list for targeted quantitative proteomics analysis using a multiple reaction monitoring (MRM) approach. To help prioritize candidate biomarkers, we constructed a protein–protein interaction network of the EPS proteins using Cytoscape (www.cytoscape.org), and overlaid the expression level changes from the Oncomine database onto the network.     

The muscle fatigue fiber model

The aim of this study was to integrate the effect of muscle fatigue into the skeletal muscle fiber model, which was previously developed in our laboratory and was based on the modified Huxley equation with the calcium activation taken into account. The cellular mechanisms such as (i) the attachment of myosin heads to the actin binding sites, (ii) the actin filament activation, (iii) the sensitivity of myofilaments to calcium ions and (iv) the calcium handling; which influence the force generated by the muscle fiber and are affected during fatigue, became the base stone for the development of the muscle fatigue fiber model.     

Analysis of the differential proteome of human erythroblasts during in vitro erythropoiesis by 2-D DIGE

In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification.     

Optical diffraction intensity of computer-simulated muscle fibers

A computer program which calculates the Fraunhofer diffraction intensity of a simulated skeletal muscle fiber is described. The intensity calculation contains several adjustable parameters. Some of the parameters are related to the myofibrillar arrangement within the muscle fiber and the angle of incident light. The variations of the left and right first-order diffraction intensities with different incident angles are determined. Their profiles exhibit features related to Bragg reflections from planes formed by the myofibrils. The calculations provide the relationship between the observed intensity profile and the myofibrillar arrangement in a real muscle fiber.     

Formation of 3-nitrotyrosines in carbonic anhydrase III is a sensitive marker of oxidative stress in skeletal muscle

Oxidation of skeletal muscle proteins has been reported to occur following contractions, with ageing, and with a variety of disease states, but the nature of the oxidised proteins has not been identified. A proteomics approach was utilised to identify major proteins that contain carbonyls and/or 3‐nitrotyrosine (3‐NT) groups in the gastrocnemius (GTN) muscles of adult (5–11 months of age) and old (26–28 months of age) wild type (WT) mice and adult mice lacking copper, zinc superoxide dismutase (Sod1^?/? mice), manganese superoxide dismutase (Sod2^+/? mice) or glutathione peroxidase 1 (GPx1^?/? mice). In quiescent GTN muscles of adult and old WT mice, protein carbonylation and/or formation of 3‐NT occurred in several proteins involved in glycolysis, as well as creatine kinase and carbonic anhydrase III. Following contractions, the 3‐NT intensity was increased in specific protein bands from GTN muscles of both adult and old WT mice. In quiescent GTN muscles from adult Sod1^?/?, Sod2^+/? or GPx1^?/? mice compared with age‐matched WT mice only carbonic anhydrase III showed a greater 3‐NT content. We conclude that formation of 3‐NT occurs readily in response to oxidative stress in carbonic anhydrase III and this may provide a sensitive measure of oxidative damage to muscle proteins.     

Motor-unit recruitment in the trapezius muscle during arm movements and in VDU precision work

It has been hypothesized that shoulder myalgia, which is common in many occupations involving light-to-moderate manual work, is due to an overuse of low threshold muscle fibers, causing damage at the muscle cell level. The present study was undertaken to investigate in the trapezius muscle, (a) to what extent it is possible to track low threshold motor-units in non-isometric conditions, and (b) to study the effect of arm movements on recruitment and firing patterns. Intramuscular four lead fine wire electrodes were inserted in the trapezius muscle of four volunteers, who performed arm movements (shoulder abduction–adduction 0–90–0° in the scapula plane), as well as standardized work using computer mouse. Three channels of electromyography (EMG) were used for motor-unit action potential (MUAP) identification and signal decomposition. Results showed (a) that it was possible to perform signal tracking during slow movements, and (b) that there were motor-units that were active over a wide range of shoulder abduction movement, as well as in work with computer mouse. The findings indicate that stereotypic recruitment patterns exist in the trapezius muscle not only in static work, but also in work situations involving arm and shoulder movements.

Relevance to industry

Occupationally related chronic shoulder pain is an increasing problem in industry, in manual work as well as in computer work. This paper presents results of studies on muscular activity patterns during movements which support the notion that chronic pain may be evoked by overuse syndromes at the muscular fiber level. Practical precautions to reduce such risks are discussed.     

Quantification of heart fatty acid binding protein as a biomarker for drug-induced cardiac and musculoskeletal necroses

Heart fatty acid binding protein (Fabp3) is a cytosolic protein expressed primarily in heart, and to a lesser extent in skeletal muscle, brain, and kidney. During myocardial injury, the Fabp3 level in serum is elevated rapidly, making it an ideal early marker for myocardial infarction. In this study, an MS‐based selected reaction monitoring method (LC‐SRM) was developed for quantifying Fabp3 in rat serum. Fabp3 was enriched first through an immobilized antibody, and the protein was digested on beads directly. A marker peptide of Fabp3 was quantified using LC‐SRM with a stable isotope‐labeled peptide standard. For six quality control samples with Fabp3 ranging from 0.256 to 25 ng, the average recovery following the procedure was about 73%, and the precision (%CV) between replicates was less than 7%. The Fabp3 concentrations in rat serum peaked 1 h after isoproterenol treatment, and returned to baseline levels 24 h after the dose. Elevated Fabp3 levels were also detected in rats administered with a PPAR α/δ agonist, which has shown to cause skeletal muscle necrosis. Fabp3 can be used as a biomarker for both cardiac and skeletal necroses. The cross‐validation of the LC‐SRM method with an existing ELISA method is described.     

Detection of protein conformation defects from fluorescence microscopy images

A diagnostic method for protein conformational diseases (PCD) from microscopy images is proposed when such conformational conflicts involve muscular intranuclear inclusions (INIs) indicative of oculopharyngeal muscular dystrophy (OPMD), one variety of PCD. The method combines two techniques: (1) the Histogram Region of Interest Fixed by Thresholds (HRIFT) is designed to capture the color information of INIs for basic feature extraction; (2) an automated feature synthesis, based on the HRIFT features, is designed to identify OPMD by means of Genetic Programming and the Expectation Maximization algorithm (GP-EM) for classification improvement. With variations in size, shape, and background structure, a total of 600 microscopic images are analyzed for the binary classes of healthy and sick conditions of OPMD. The integrated technique of the approach reveals a sensitivity of 0.9 and an area of 0.961 under the receiver operating characteristic (ROC) at a specificity of 0.95. Furthermore, significant improvements in classification accuracy and computational time are demonstrated by comparison with other methods.     

The specificity of endurance training on muscular power and muscle fibre size

In an effort to further examine the specificity of training concept, six female student volunteers (mean age=25.3 years) were endurance trained three times a week for a period of twelve weeks. The training consisted of thirty minutes of continuous running per workout. Identical test batteries were administered prior to (pre) and immediately following (post) the treatment period. Each battery of tests consisted of three measures of muscular leg power (vertical jump test, 18-29 m sprint, and the Margaria Step Test), and two measures of cardiovascular endurance (Cooper's 12 minute run, and V?O₂ max during treadmill running). A muscle biopsy from the lateral aspect of the gastrocnemius muscle was also included. The biopsy specimens were stained for myosin ATPase. Percentages of fast twitch (FT) and slow twitch (ST) muscle fibres as well as mean individual FT and ST fibre areas were determined. The training produced significant changes in the exercising subjects for thel2-minute run (p=0.006)and VO₂max(p=0.003). No changes were found in the tests of muscular power or individual fibre areas. These data indicate that the endurance training program was of sufficient intensity to effect an increase in cardiovascular fitness. However, these improvements in endurance were not accompanied by any significant changes in muscular leg power or individual muscle fibre size.     

2-D DIGE identification of differentially expressed heterogeneous nuclear ribonucleoproteins and transcription factors during neural differentiation of human embryonic stem cells

Neural stem cells (NSC) are progenitors that can give rise to all neural lineages. They are found in specific niches of fetal and adult brains and grow in vitro as non-adherent colonies, the neurospheres. These cells express the intermediate filament nestin, commonly considered an NSC marker. NSC can be derived as neurospheres from human embryonic stem cells (hESC). The mechanisms of cellular programming that hESC undergo during differentiation remain obscure. To investigate the commitment process of hESC during directed neural differentiation, we compared the nuclear proteomes of hESC and hESC-derived neurospheres. We used 2-D DIGE to conduct a quantitative comparison of hESC and NSC nuclear proteins and detected 1521 protein spots matched across three gels. Statistical analysis (ANOVA n = 3 with false discovery correction) revealed that only 2.1% of the densitometric signal was significantly changed. The ranges of average ratios varied from 1.2- to 11-fold at a statistically significant p-value <0.05. MS/MS identified 15 regulated proteins previously shown to be involved in chromatin remodeling, mRNA processing and gene expression regulation. Notably, three members of the heterogeneous nuclear ribonucleoprotein family (AUF-1, and FBP-1 and FBP-2) register a 54, 70 and 99% increased expression, highlighting them as potential markers for NSC in vitro derivation. By contrast, Cpsf-6 virtually disappears with differentiation with an 11-fold drop in NSC, highlighting this protein as a novel marker for undifferentiated ESC.     

Biohybrid device with antagonistic skeletal muscle tissue for measurement of contractile force

ABSTRACT

This paper describes a fabrication method and driving property of a biohybrid device with an antagonistic pair of skeletal muscle tissues and a flexible substrate. Since two skeletal muscle tissues are symmetrically arranged with the flexible substrate as the central axis, the flexible substrate deforms according to differences in their tension. In the formation of the antagonistic pair of skeletal muscle tissues, we assembled myoblast-laden hydrogel sheets and cultured them to construct a single skeletal muscle tissue on each side of the flexible substrate. We confirmed that the skeletal muscle tissue on the flexible substrate had the fundamental morphology and function of skeletal muscle. Furthermore, we made the biohybrid device actuate with deformation of the flexible substrate by selective contractions of the skeletal muscle tissues. From the deformation of the flexible substrate, we estimated the contractile force of each skeletal muscle tissue in the biohybrid device using finite element analysis. This biohybrid device, composed of an antagonistic pair of skeletal muscle tissues and a flexible substrate, can potentially be used in biological studies and pharmacokinetic assays involving the antagonistic pair of skeletal muscles.